Diagnostic Tests
Diagnostic Tests
This section has been designed to assist you with understanding the use of
the diagnostic tests used in immunological testing and clinical practice. The
use of these tests is explained with the aid of easy to follow colour graphics
and text. The application of these tests is featured in our various case
studies which are linked to this section giving you a quick reference while
working through the cases.
If you would like to have a specific diagnostic test featured please Contact Us.
Alere HIV Combo - Rapid Test
ELISA tests
Lymphocyte Proliferation Test
PCR test
Mantoux test
Viral Load Assays
Flow Cytometry
Hepatitis B Surface Antigen
Rapid HIV Test
Ouchterlony test
CD4 Count
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Alere HIV Combo
[Alere.com]
Alere™ HIV Combo – Rapid Test
in vitro
Qualitative immunoassay
4th generation Rapid Test
Detection of antibodies (Ab) to HIV-1 and HIV-2
Detection of free HIV-1 p24 antigen (Ag)
As it detects p24 antigens, it may identify HIV infection earlier than antibody-only tests
The multiplication of the HIV in the infected cells leads to cell rupture and thus the release of HIV virus
particles, which are first detected in the form of HIV RNA and next in the form of HIV antigen. This is
followed by production of specific antibodies to either HIV-1 or HIV-2. The HIV antigen may become
undetectable at this time because of the formation of antibody-antigen complexes. Alere HIV Combo
can only detect free p24 antigens.
Test Procedure
Rapid and easy-to-use
Lateral flow assay
50 μL of sample applied to a sample pad
1 drop of Chaser Buffer applied to sample pad
Sample diffuses along the strip
Results for HIV-1 p24 antigen and HIV-1 and HIV-2 antibodies
Control window
Results read in designated windows
The small strip format is suitable for use with whole blood, serum, or plasma samples
Results in 20-40 minutes
Alere HIV Combo test procedure [Alere HIV Combo REF 7D2842, 7D2843 – June 2016, 241336/R4]
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Test Results
Control and Ab bars – Antibody reactive
Control and Ag bars – Antigen p24 reactive
Control, Ab and Ag bars – Antibody and Antigen p24 reactive
Only Control bar – Non-reactive
No Control bar – Test invalid
Alere HIV Combo test results [Alere HIV Combo REF 7D2842, 7D2843 – June 2016, 241336/R4]
How the Test Works
A specimen is added to the sample pad.
The specimen mixes with biotinylated anti-p24 antibodies and selenium colloid-conjugates
coated with recombinant HIV-1, HIV-2 and HIV-1 group O antigens, synthetic HIV-2 peptide and
anti p24 mouse monoclonal antibody.
This mixture continues to migrate through the solid phase to the immobilized recombinant
HIV-1/HIV-1 group O antigens and synthetic HIV-1/HIV-2 peptides at the Antibody (Ab) window,
immobilized avidin at the Antigen (Ag) window.
If antibodies to HIV-1 and/or HIV-2 are present in the specimen, the antibodies bind to the
selenium colloid-conjugates coated with recombinant HIV-1, HIV-2 and HIV-1 group O antigens
and synthetic HIV-2 peptide and to the immobilized recombinant HIV-1/HIV-1 group O antigens
and synthetic HIV-1/HIV-2 peptides, forming one red bar at the Ab window site.
If antibodies to HIV-1 and HIV-2 are absent, the selenium colloid-conjugates flow past the Ab
window and no red bar is formed at the Ab window site.
If free HIV-1 p24 antigen is present in the specimen, the antigen binds to the biotinylated antip24 antibodies and the selenium colloid-conjugate coated with anti p24 mouse monoclonal
antibody. This complex binds to the immobilized avidin forming a red bar at the Ag window site.
If HIV-1 p24 antigen is not present, both the biotinylated anti-p24 antibodies and selenium
colloid conjugate flow past the Ag window and no red bar is formed at the Ag window site.
To ensure assay validity, a procedural control bar is incorporated in the assay device at the
Control window.
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Test Limitations
No test provides absolute assurance that a specimen does not contain low levels of HIV-1 p24
antigen and/or
antibodies to HIV-1 and HIV-2 such as those present at a very early stage or late stage of HIV
infection.
Alere HIV Combo is designed to detect antibodies to HIV-1 and/or HIV-2 and free HIV-1 p24
antigen, in human serum, plasma and capillary and venous whole blood specimens. Other body
fluids or pooled specimens should not be used.
The intensity of the Ab and Ag bars does not necessarily correlate to the titer of antibody and
antigen in the specimen.
The absence of Ag bar may occur when all p24 antigen is bound by antibodies. When high
levels of antibodies against the p24 antigen are present in the blood after seroconversion, the
antibodies tend to bind to the antigens, forming immunocomplexes. Alere HIV Combo detects
only non-immunocomplexed (free) antigens; it does not detect immunocomplexed (bound)
antigens.
HIV-infected persons taking antiretroviral medication have been shown to produce false
negative results when tested by rapid diagnostic tests.
Infants born to HIV-infected mothers may carry maternal antibodies and will test antibody
positive until eighteen months of age, which may not necessarily indicate the true infection
status of the new born. The use of HIV- 1 p24 antigen testing to exclude infection in neonates
(up to around eighteen months) is not recommended by CDC, because of poor sensitivity,
especially in the presence of HIV antibody. Definitive diagnosis of HIV infection in early infancy
requires other assays, including HIV nucleic acid test or viral culture.
More Information
Alere.com – Alere HIV Combo
ELISA tests
ELISA (enzyme –linked immunosorbent assay) is a serological assay in which a bound antigen or
antibody is detected by a linked enzyme that converts a colourless substrate into a coloured product.
There are 4 generations of HIV antibody ELISA tests. The progression of these assays has been
depicted in our series of 4 graphics.
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Lymphocyte Proliferation Test
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The Lymphocyte Proliferation Assay (LPA) is a test used
to measure the ability of lymphocytes to proliferate in
response to various stimuli such as candida, pokeweed
and phytohaemagglutinin. These plant lectins are
carbohydrate binding proteins that bind to cell surface
receptors and activate cells in an antigen independent
manner. Most people will respond to at least one of
several common microbial antigens. LPA can be used to
measure improvements in immunological function
following antiretroviral therapy and to detect the
presence of immune responses against specific
opportunistic pathogens.
The 3H-thymidine incorporation assay is used to determine the extent of cell division in response to a
proliferation signal. 3H-thymidine which is a radioactive nucleoside and precursor of thymine found in
DNA becomes incorporated in DNA strands of proliferating cells and can be measured to determine
extent of cell proliferation.
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PCR test
The polymerase chain reaction (PCR) is a technique used
for a wide range of tasks including detection and
diagnosis of infectious disease. The technique
exponentially amplifies a fragment of DNA or RNA by in
vitro enzymatic replication and because PCR amplifies
the regions of DNA or RNA that it targets, PCR can be
used to analyze extremely small amounts of sample.
Viral DNA or RNA can therefore be detected by PCR. The
primers used need to be specific to the targeted
sequences in the DNA or RNA of a virus, and the PCR
can be used for diagnostic analyses or DNA or RNA
sequencing of the viral genome. The high sensitivity of
PCR permits virus detection soon after infection and even before the onset of disease, allowing for a
significant lead in treatment. A patient’s viral load can also be quantified by PCR-based DNA or RNA
quantitation techniques.
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Mantoux test
The Mantoux test also called the Purified Protein Derivative (PPD test) or Tuberculin Sensitivity Test is
a diagnostic tool for tuberculosis. The test shows if a person has been exposed to TB bacteria, but it
does not indicate if the infection is active or latent or if the patient is currently infectious. This test is
an example of a Type IV or cell-mediated/delayed hypersensitivity immune response.
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Viral Load Assays
The viral load assay is used to monitor the status of HIV disease, in conjunction with the CD4 count
and physical disease progression. It is also used to guide therapy and to predict the progression of
HIV. A viral load is conducted when a patient is first diagnosed with HIV, at the start of therapy or
when a therapy is changed and regularly thereafter to monitor any changes. With effective therapy,
viral loads typically decrease within 3-6 months. When a patient has <25 copies of virus they are said
to be virally suppressed. Evidence shows that keeping the viral load levels as low as possible for as
long as possible decreases the complications of HIV disease.
There are several methods for testing viral load; results are not interchangeable so it is important that
the same method be used each time.
The 5 methods for testing Viral Load are:
a) Roche Amplicor
b) Versant Branched DNA
c) Roche COBAS taqman
d) Nuclisens
e) Abbot HIV real-time quantification
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Flow Cytometry
Manual methods using cell selection and microscope
counting techniques can be used, however, automated
counting of CD4+ Helper T lymphocytes in blood is
usually performed on a flow cytometer. Although more
expensive, this method is more accurate and less labour
intensive.
The flow cytometer uses fluorescent cell surface markers (usually these are labelled antibodies) to
identify the subset of CD4+ Helper T lymphocytes present in blood samples.
There are two approaches regarding the type of cell surface markers used.
1) Conventional CD4 counting (CD3 and CD4 labelling)
The conventional method makes use of fluorescent antibodies to CD3 and CD4 cell surface receptors.
CD3 is part of the T cell receptor (TCR) and is expressed on CD8+ Cytotoxic T lymphocytes and CD4+
Helper T lymphocytes. CD4 is expressed on Helper T lymphocytes and monocytes. Using fluorescence
detection on a flow cytometer the fraction of total lymphocytes double-positive for CD3 and CD4 (i.e.
CD4+ Helper lymphocytes) can be measured. For single platform assays the absolute number of the
double-positive cells can be inferred from calibrated bead standards or by determining the volume of
blood used with a volumetric flow cytometer. The dual-platform method relies on a full blood count by
a haematology analyser to provide a value for the total lymphocyte count, while the flow cytometer
gives a percentage of lymphocytes that are CD4+ Helper T lymphocytes. Together these readings can
be used to calculate absolute CD4 counts. The most reliable and robust counting procedure is the
single platform assay.
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2) CD4 counting by the PanLeucogate method (CD45 and CD4 labelling)
An alternative labelling method uses fluorescent antibodies to CD45 and CD4 cell surface receptors.
CD45 is expressed on all leucocytes and is not expressed on mature red blood cells (erythrocytes) or
platelets (thrombocytes). It therefore serves as a marker of total white blood cells. CD4 is expressed
on Helper T lymphocytes and monocytes. The dual platform method requires a haematology analyzer
to provide only the total leukocyte count. This is the reason PanLeucogating is preferred when using
the dual platform method, only a total white cell count is needed from the haematology analyzer and
the need for a differential count (the lymphocyte count) from the haematology analyser is no longer
necessary. The leucocytes serve as a common counting parameter when using the flow cytometer
and haematology analyzer. Since CD4 is expressed on both Helper T lymphocytes and monocytes the
light scatter parameters available on the flow cytometer are used to differentiate between them
because of differences in complexity between the populations. This method can also be used on a
single platform basis using calibrated bead standards or a volumetric flow cytometer to calculate the
absolute CD4 count.
Hepatitis B Surface Antigen
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Hepatitis B surface antigen (HBsAg) is a protein antigen
produced by hepatitis B virus (HBV). This antigen is the earliest indicator of acute hepatitis B infection
and is able to identify infected people before they are symptomatic. HBsAg becomes undetectable in
the blood during the recovery period. Children and immunocompromised patients such as patients
with HIV infection who become chronically infected may remain HBsAg positive.
To detect this antigen we perform an Enzyme-Linked Immunosorbent Assay test (HIV ELISA).
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A baby born to a sero-positive mother will also be seropositive. This positivity can last up to the first 18 months
of life because the test detects maternal antibodies. For
this reason, it is useless to test the baby at two months
using an HIV antibody test as IgG will have either
crossed the placenta to the baby in utero or via beast
milk postnatal. Until these antibodies disappear the
baby will continue to test sero-positive. After 18
months, if the baby is truly infected with HIV, the child’s
own immune system will be the cause of a sero-positive
antibody test.
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Ouchterlony test
Immune precipitates can form in an agar matrix. When antigen and antibody diffuse toward one
another in agar, or when antibody is incorporated into the agar and antigen diffuses into the antibody
containing matrix, a visible line of precipitation will form. As in a precipitation reaction in fluid, visible
precipitation occurs in the region of equivalence, whereas no visible precipitate forms in regions of
antibody or antigen excess. This immunodiffusion reaction can be used to determine relative
concentrations of antibodies or antigens, to compare antigens or to determine the relative purity of
an antigen preparation.
In the Ouchterlony method both antigen and antibody diffuse radially from wells toward each other,
thereby establishing a concentration gradient. As equivalence is reached, a visible line of precipitation
forms. This is a simple and effective qualitative tool for determining the relationship between antigens
and the number of different Ag-Ab systems present.
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CD4 Count
In this series of graphics the three basic cell types found
in blood (leukocytes, erythrocytes and platelets) have
been depicted. The graphics further explain the
leukocyte cell lines and include the granulocytes,
monocytes and lymphocytes and the different cells
which make up these groups of cells.
When measuring disease states it is an absolute CD4 count which is used to monitor patients.
Although CD4 cell counts are used to measure HIV disease states absolute CD4 counts are not a
reliable marker when determining disease states in children. This is because lymphocytes are present
in higher numbers in children thus making the count less
accurate. The WHO has therefore recommended that all
children under the age of 5 should be evaluated with a
CD4 percentage when assessing disease status and
decision making for initiation of treatment.
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